基于DNA条形码和PCR-RFLP技术的进口紫草ITS2序列特征研究

The ITS2 sequences’characters study of imported Arnebiae Radix based on DNA barcoding and PCR-RFLP technologies

目的:基于DNA条形码和PCR-RFLP技术研究进口紫草ITS2序列的特征,为市场紫草药材和饮片的质量控制与真伪鉴别提供参考依据。方法:选用ITS2区域作为对进口紫草和紫草对照药材进行比较、鉴定的DNA条形码序列,并基于DNA条形码和PCR-RFLP技术比较不同来源进口紫草的ITS2序列与紫草对照药材的异同。结果:39份进口紫草样品经限制性内切酶AluI酶切后,其产物的琼脂糖凝胶电泳检测结果显示,仅DH3在500 bp左右有条带,而在100~300 bp无条带,其余样品均在100~300 bp有2条或3条明显条带;进口紫草样品与紫草对照药材的ITS2序列进行比对,其中样品DH3与紫草对照药材的碱基差异最多,有15个碱基差异,样品F2与紫草对照药材的ITS2序列一致,其他进口紫草样品与紫草对照药材的碱基差异为1~9个碱基;从聚类结果中可以看出进口紫草样品DH3与其他进口紫草样品和紫草对照药材均明显区分,独自为一枝,而与紫草对照药材共同聚为一枝且支持率≥50%的样品共有14个。结论:选用ITS2区域,基于DNA条形码和PCR-RFLP技术,比较了进口紫草与紫草对照药材ITS2序列的异同,为紫草药材及饮片的有效鉴定提供参考依据,为紫草药材的市场监管提供有力保障。

Objective:To provide reference for quality control and authenticity identification of Arnebiae Radix medicinal materials and decoction pieces in the market. By studied on the ITS2 sequences' characters of imported Arnebiae Radix, based on DNA barcoding and PCR-RFLP technologies. Methods: The ITS2 region was selected as the DNA barcode sequence for comparison and identification of imported Arnebiae Radix and reference medicinal materials. The ITS2 sequences of imported Arnebiae Radix from different sources with reference medicinal materials were compared based on DNA barcoding and PCR-RFLP technologies. Results: After the restriction endonucliase AluI enzyme digestion, the agarose-gel electrophoresis results of 39 imported Arnebiae Radix samples showed that, only DH3 had bands at around 500 bp, and none bands between 100 bp and 300 bp. And the results of other imported Arnebiae Radix samples had two or three obvious bands between 100 bp and 300 bp. The ITS2 sequences of imported Arnebiae Radix samples were compared with the reference medicinal materials, among which DH3 had the largest differences of 15 bases compared to the reference medicinal materials, the ITS2 sequence of F2 was same to the reference medicinal materials, and other imported Arnebiae Radix samples had 1-9 bases difference compared to the reference medicinal materials. The clustering results showed that the imported Arnebiae Radix sample DH3 was clearly distinguished from other imported Arnebiae Radix samples and reference medicinal materials which was in a single branch. There were 14 samples, which were clustered together with the reference medicinal materials in one branch with support rate ≥50%. Conclusion: The ITS2 region is selected to compare the similarities and differences of ITS2 sequences between imported Arnebiae Radix samples and reference medicinal materials based on DNA barcode and PCR-RFLP technologies, which provids a reference for effective identification of Arnebiae Radix medicinal materials and decoction pieces, and a strong guarantee for market supervision of Arnebiae Radix medicinal materials.